Preparation Andidentification Of Monoclonal Antibodies Against Luman-N-Term Fusion Protein

Luman is a cellular transcription factor that associtated with herpes simplex virus infected and reactivate from latenrancy. Luman is one of basic domain leucine zipper transcription factors. It is one of transcription factors participating regulation in endoplasmic reticulum stress and unfolded protein response. Its molecular structure is divided into two departments, in which N-term region is associtated with the regulation function of transcription .The Luman-N-term region molecular weight probably is 30 ku.All of Luman known functional regions including active domain, host cell factor-binding motifs and basic leucine zipper region are all allocated in N-term of Luman.Its research is momentous to explain the regulation mechanism of Luman.E. coli with Luman-N-term Fusion gene was induced by IPTG, and identified by SDS-PAGE then the expression condition was optimized. The expressed fusion protein was purified by electrical elution. The purified Luman-N-term fusion protein was used to immune BALB/C mice. The titer of anti-Luman-N-term fusion protein serum was tested by indirectly ELISA. The immunitied mice spleen cells and SP2/0 myelomas cells were fused under the working of 50 % PEG 4000 and the positive clone was screened by limiting dilution method. Then the McAbs were prepared and identificated. The results indicated that:1. The results showed that the highly expressed Luman-N-term fusion protein ,which mainly existed in the inclusion body ,was obtained in the optimal condition of 37℃,1 mmol/ L of IPTG and induction for 7 h.The Bradford Protein Assay determination indicated that the recombinant protein concentration was about 0.65 mg/mL.2. Indirect ELISA method is established: HRP-labelled goat anti mouse IgG diluted by PBST (pH 7.4, 0.1 mol/L) in l∶1500 was choosed as secondary antibody, coated antigen 5μg/mL Luman-N-term fusion protein dissolved in carbonate buffer solution (CBS, pH 9.6, 0.05 mol/L) for an hour and a night at 37℃, 1.0 % gelatinum as blocking agent, and action time of enzyme labelled antibody was 30 min .3 Splenic cells of Balb/c injected were hybridized with SP2/0 by cell fusion and the hybridoma cells were screened by ELISA and subcloned approach. Finally three hybridoma cells (8B2G,3A5E and 1A3F) stably secreting anti-Luman-N-term fusion protein monoclonal antibodies were obtained, and ascites were induced to produce monoclonal antibodies. Hybridoma cells and the monoclonal antibodies were identified ultimately. The results indicated that hybridoma cells were stably secreting antibodies after freezing and thawing and passage culture. The McAbs showed high titer to purified Luman-N-term fusion protein with various affinity. Western-blot indicated that three strain McAbs presented high specificity to Luman-N-term fusion protein expressed in the prokaryotic cells. The cross-reaction rate of McAb to Zhangfei fusion protein and LRF fusion protein was all below 0.5 %.In the end, we produced anti-Luman-N-term fusion protein monoclonal antibodies successfully, which were foundation for the further study on function of Luman.